5989-9665EN_EPA Method 1694

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EPA Method 1694: Agilent's 6410ALC/MS/MS Solution for

Pharmaceuticals and Personal CareProducts in Water, Soil, Sediment,and Biosolids by HPLC/MS/MS

Abstract

An analytical methodology for screening and confirming the presence of 65 pharma-

ceuticals in water samples was developed using the Agilent G6410A Triple

Quadrupole mass spectrometer (QQQ). The method was developed following the

guidelines in EPA Method 1694. Four distinct chromatographic gradients and LC con-

ditions were used according to the polarity and extraction of the different pharmaceu-

ticals. Positive and negative ion electrospray were used with two multi-reaction moni-

toring (MRM) transitions (a quantifier and a qualifier ion for each compound), which

adds extra confirmation in this methodology compared with the EPA method. Linearity

of response of three orders of magnitude was demonstrated (r2 > 0.99) for all the

pharmaceuticals studied. The analytical performance of the method was evaluated for

one wastewater sample collected from Boulder Creek, Colorado; positive identifica-

tions for carbamazepine and diphenhydramine were found for this sample using the

methodology developed in this work.

Authors

Imma Ferrer and E. Michael Thurman

Center for Environmental Mass

Spectrometry

University of Colorado

Civil, Environmental, and Architectural

Engineering

ECOT 441, 428 UCB

Boulder, CO 80309

USA

Jerry Zweigenbaum

Agilent Technologies, Inc.

2850 Centerville Road

Wilmington, DE 19808

USA

Application Note

Environmental

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Introduction

The analytical challenge of measuring emerging contaminants

in the environment has been a major research focus of scien-

tists for the last 20 years. Pharmaceuticals and personal care

products (PPCPs) are an important group of contaminants

that have been targeted, especially in the last decade. In the

area of PPCPs there are several methods addressing the

analysis of these analytes, including EPA Method 1694 [1],

which was recently published (December 2007). This EPA pro-

tocol uses solid-phase extraction (SPE) for water sample

preparation [1]. The extracts are then analyzed directly by a

tandem mass spectrometer using a single transition for each

compound. This application note describes the Agilent solu-

tion to this method, which is demonstrated with the Agilent

model 6410A LC/MS QQQ. The Agilent initial implementationfor EPA Method 1694 consists of 65 analytes (of 75 total ana-

lytes) and 17 labeled internal standards (of 20 total), which

are a mixture of PPCPs that are analyzed each by a single

MRM transition. (Note that the other compounds and internal

standards could not be obtained at this time.) The method

also uses Agilent C-18 and Hydrophilic Interaction

Chromatography (HILIC) columns for all analytes. To provide

additional confirmation, a second MRM transition was added

for 60 of the 65 analytes analyzed. This gives an even greater

assurance of correct identification than prescribed by the

EPA. Table 1 shows the list of pharmaceuticals studied here.

Table 1. Analytes Studied in This Work

Acetaminophen Codeine Flumequine Penicillin V Sulfanilamide

Ampicillin Cotinine Fluoxetine Roxithromycin Thiabendazole

Azithromycin Dehydronifedipine Lincomycin Sarafloxacin Trimethoprim

Caffeine Digoxigenin Lomefloxacin Sulfachloropyridazine Tylosin

Carbadox Diltiazem Miconazole Sulfadiazine Virginiamycin

Carbamazepine 1,7-Dimethylxanthine Norfloxacin Sulfadimethoxine Digoxin*

Cefotaxime Diphenhydramine Ofloxacin Sulfamerazine

Ciprofloxacin Enrofloxacin Oxacillin Sulfamethazine

Clarithromycin Erythromycin Oxolinic acid Sulfamethizole

Cloxacillin Erythromycin anhydrate Penicillin G Sulfamethoxazole*Compound formed intractable Na adduct with current conditions.

List of Group 1 Compounds EPA 1694: 46 Analytes

List of Group 2, 3, and 4 Compounds: EPA 1694: 19 Analytes

Anhydrotetracycline (2) Doxycycline (2) Minocycline (2) Triclocarban (3)

Triclosan (3)

Warfarin (3)

Chlorotetracycline (2) 4-Epianhydrotetracycline (2) Tetracycline(2) Albuterol (4)

Meclocycline (2) Cimetidine (4)

Metformin (4)

Demeclocycline(2) 4-Epitetracycline(2) Gemfibrozil (3) Ranitidine (4)

Ibuprofen (3)

Naproxen (3)

List of Labeled Internal Standards

13C2-15N-Acetaminophen 13C2-Erythromycin

13C6-Sulfamethazine13C3-Trimethoprim

13C3-Atrazine Fluoxetine-d613C6-Sulfamethoxazole Warfarin-d5

13C3-Caffeine Gemfibrozil-d613C6-2,4,5-Tricloro- Carbamazepine-d10phenoxyacetic acid (Extra compound, not EPA list)

13C3-15N-Ciprofloxacin 13C3-Ibuprofen

13C6-Triclocarban

Cotinine-d313C-Naproxen-d3

13C12-Triclosan

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Experimental

Sample Preparation

Pharmaceutical analytical standards were purchased from

Sigma, (St. Louis, MO). All stable isotope labeled compounds

used as internal standards were obtained from Cambridge

Isotope Laboratories (Andover, MA). Individual pharmaceuti-

cal stock solutions (approximately 1,000 µg/mL) were pre-

pared in pure acetonitrile or methanol, depending on the solu-

bility of each individual compound, and stored at

–18 °C. From these solutions, working standard solutions

were prepared by dilution with acetonitrile and water.

Water samples were collected from the wastewater treat-

ment plant at the Boulder Creek outfall (Boulder, CO) and

extracted as per the EPA method. Agilent has introduced apolymeric SPE sorbent with hydrophilic/lipophilic properties

that may also be appropriate for this application. “Blank”

wastewater extracts were used to prepare the matrix-

matched standards for validation purposes. The wastewater

extracts were spiked with the mix of pharmaceuticals at dif-

ferent concentrations (ranging from 0.1 to 500 ng/mL or ppb)

and subsequently analyzed by LC/MS/MS.

LC/MS/MS Instrumentation

The analytes were subdivided in groups (according to EPA

protocol for sample extraction) and LC conditions for the

chromatographic separation of each group are as follows.

LC Conditions for Group 1-acidic extraction, positive

electrospray ionization (ESI+) instrument conditions

Column Agilent ZORBAX Eclipse Plus C18

2.1 × 100 mm, 3.5 µ (p/n 959793-902)

Column temperature 25 °C

Mobile phase 10% ACN and 90% H2O with 0.1% HCOOH

Flow rate 0.2–0.3 mL/min

Gradient t0 = 10% ACN, 0.2 mL/min

t5 = 10% ACN, 0.2 mL/min

t6 = 10% ACN, 0.3 mL/min

t24 = 60% ACN, 0.3 mL/mint30 = 100% ACN

Injection volumes 15 µL

LC conditions for Group 2-acidic extraction, positive electrospray

ionization (ESI+) instrument conditions

Column Agilent ZORBAX Eclipse Plus C182.1 × 100 mm, 3.5 µ (p/n 959793-902)


Mobile phase 10% ACN and 90% H2O with 0.1% HCOOH

Flow rate 0.2 mL/min

Gradient t0 = 10% ACN

t10 = 10% ACN

t30 = 100% ACN


LC conditions for Group 3-acidic extraction, negative electrospray

ionization (ESI–) instrument conditions

Column Agilent ZORBAX Eclipse Plus C18

2.1 × 100 mm, 3.5 µ (p/n 959793-902)


Mobile phase 40% MeOH and 60% H2O with

5 mM ammonium acetate, pH 5.5


Gradient t0.5 = 40% MeOH

t7 = 100% MeOH


LC conditions for Group 4-acidic extraction, positive electrospray

ionization (ESI+) instrument conditions

Column Agilent ZORBAX HILIC Plus

2.1 × 100 mm, 3.5 µm (p/n 959793-901

custom order until November 1, 2008)


Mobile phase 98% ACN and 2% H2O with 10 mM

ammonium acetate, pH 6.7


Gradient t0 = 98% ACN

t5 = 70% ACN

t12 = 70% ACNInjection volumes 15 µL

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The mass spectrometer conditions were general to all groups

and are as follows.

MS ConditionsMode Positive and negative (depending on

group) ESI using the Agilent G6410A

Triple Quadrupole mass spectrometer

Nebulizer 40 psig

Drying gas flow 9 L/min

V capillary 4000 V

Drying gas temperature 300 °C

Fragmentor voltage 70–130 V

Collision energy 5–35 V

MRM 2 transitions for every compound as shown

in Table 1

Dwell time 10 msec

Results and Discussion

Optimization of LC/MS/MS Conditions

The initial study consisted of two parts. First was to optimize

the fragmentor voltage for each of the pharmaceuticals stud-

ied in order to produce the largest signal for the precursor ion.

Typically the protonated molecule was used for the precursor

ion. Each compound was analyzed separately using an auto-

mated procedure (MassHunter Optimizer software, Agilent

Technologies, Santa Clara, CA) to check the fragmentor at

each voltage. The data was then selected for optimal frag-

mentor signal and each compound was optimized again to

determine automatically the collision energies for both the

quantifying and qualifying ions. Optimal collision energies var-

ied between 5 and 35 V. The MRM transitions and optimized

energies used for this study are shown in Tables 2A to 2D.

Table 2A. MRM Transitions and MS Operating Parameters Selected for the Analysis of the Pharmaceutical Compounds in Group 1 (The

labeled standards are bold.)

Fragmentor MRM Collision energy

Compound voltage transitions (m/z ) (eV)

Acetaminophen 90 152→ 110 15

152→ 65 35

13C2-15N-Acetaminophen 90 155→ 111 15

155→ 93 25

Ampicillin 70 350→ 160 10350→ 106 15

13C3-Atrazine 120 219→ 177 15

219→ 98 25

Azithromycin 130 749.5→ 591.4 30

749.5→ 158 35

Caffeine 110 195→ 138 15

195→ 110 25

13C3-Caffeine 110 198→ 140 15

198→ 112 25

Carbadox 80 263→ 231 5

263→ 130 35

Carbamazepine 110 237→ 194 15

237→ 179 35

Carbamazepine-d10 110 247→ 204 15

247→ 202 35

Cefotaxime 90 456→ 396 5

456→ 324 5

Ciprofloxacin 110 332→ 314 20

332→ 231 35

13C3-15N-Ciprofloxacin 110 336→ 318 15

336→ 235 35

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Clarithromycin 110 748.5→ 158 25

748.5→ 590 15

Cloxacillin 90 436→ 160 15

436→ 277 15

Codeine 130 300→ 215 25

300→ 165 35

Cotinine 90 177→ 98 25

177→ 80 25

Cotinine-d3 90 180→ 80 25

180→ 101 25

Dehydronifedipine 130 345→

284 25345→ 268 25

Digoxigenin 90 391→ 355 15

391→ 337 15

Digoxin No response, Na adduct

Diltiazem 130 415→ 178 25

415→ 150 25

1,7-Dimethylxanthine 90 181→ 124 15

181→ 99 15

Diphenhydramine 70 256→ 167 15

256→ 152 35

Enrofloxacin 130 360→ 316 15

360→ 342 15Erythromycin 90 734.5→ 158 35

734.5→ 576 15

13C2-Erythromycin 90 736.5→ 160 25

736.5→ 578 15

Erythromycin anhydrate 90 716.5→ 158 25

716.5→ 116 25

Flumequine 90 262→ 174 35

262→ 244 15

Fluoxetine 90 310→ 148 5

Fluoxetine-d6 90 316→ 154 5

Lincomycin 110 407→ 126 25

407→ 359 15

Lomefloxacin 130 352→ 308 15

352→ 265 25

Miconazole 90 415→ 159 35

415→ 69 25

Norfloxacin 70 320→ 302 15

320→ 276 15

Ofloxacin 110 362→ 318 15

362→ 261 25



Table 2A. MRM Transitions and MS Operating Parameters Selected for the Analysis of the Pharmaceutical Compounds in Group 1

(The labeled standards are bold.) continued

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Oxacillin 70 402→ 160 15

402→ 243 5

Oxolinic acid 90 262→ 244 15

262→ 216 25

Penicillin G 90 335→ 160 5

335→ 176 5

Penicillin V 70 351→ 160 5

351→ 114 25

Roxithromycin 130 837.5→ 679 15

837.5→ 158 35

Sarafloxacin 130 386→ 299 25

386→ 368 25

Sulfachloropyridazine 90 285→ 156 10

285→ 92 25

Sulfadiazine 110 251→ 156 15

251→ 92 25

Sulfadimethoxine 80 311→ 156 20

311→ 92 35

Sulfamerazine 110 265→ 156 15

265→ 92 25

Sulfamethazine 90 279→ 156 15

279→ 186 15

13C6-Sulfamethazine 90 285→ 186 25

285→ 162 25

Sulfamethizole 80 271→ 156 10

271→ 92 25

Sulfamethoxazole 110 254→ 156 15

254→ 92 25

13C6-Sulfamethoxazole 110 260→ 162 15

260→ 98 25

Sulfanilamide 70 173→ 156 5

173→ 92 15

Thiabendazole 130 202→ 175 25

202→ 131 35

13

C6-2,4,5-Trichlorophenoxyacetic acid 110 259→

201 5259→ 165 25

Trimethoprim 110 291→ 230 25

291→ 261 25

13C3-Trimethoprim 110 294→ 233 25

294→ 264 25

Tylosin 110 916.5→ 174 35

916.5→ 772 35

Virginiamycin 110 526→ 508 5

526→ 355 15



Table 2A. MRM Transitions and MS Operating Parameters Selected for the Analysis of the Pharmaceutical Compounds in Group 1 (The

labeled standards are bold.) continued

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Table 2B. MRM Transitions and MS Operating Parameters Selected for the Analysis of the Pharmaceutical Compounds in Group 2

Anhydrotetracycline 90 427→ 410 15

427→ 154 25

Chlorotetracycline 110 479→ 462 15

479→ 197 35

Demeclocycline 130 465→ 430 25

465→ 448 15

Doxycycline 110 445→ 428 15

445→ 154 25

4-Epianhydrotetracycline (EATC) 90 427→ 410 15

427→ 105 35

4-Epitetracycline (ETC) 110 445→

410 15445→ 427 5

Minocycline 90 458→ 441 15

Tetracycline (TC) 110 445→ 410 15

445→ 427 5



Table 2C. MRM Transitions and MS Operating Parameters Selected for the Analysis of the Pharmaceutical Compounds in Group 3



Gemfibrozil 100 249→ 121 5

Gemfibrozil-d6 100 255→ 121 5

Ibuprofen 75 205→ 161 513C3-Ibuprofen 75 208→ 163 5

Naproxen 75 229→ 169 25

229→ 170 5

13C-Naproxen-d3 75 233→ 169 25

233→ 170 5

Triclocarban 100 313→ 160 10

313→ 126 25

13C6-Triclocarban 90 319→ 160 5

319→ 132 25

Triclosan 75 287→ 35 5

13C12

-Triclosan 75 299→ 35 5

Warfarin 125 307→ 117 35

307→ 161 15

Warfarin-d5 90 312→ 161 15

312→ 255 25

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Chromatographic separation was done independently for each

group and a dwell time of 10 msec was used for every MRM

transition. Figures 1A to 1D show the chromatograms corre-sponding to 100 ppb standard on column for all the pharma-

ceuticals studied. Extracted ion chromatograms are overlaid

for each one of the target analytes according to their respec-

tive protonated molecule and product-ion MRM transitions.

Table 2D. MRM Transitions and MS Operating Parameters Selected for the Analysis of the Pharmaceutical Compounds in Group 4



Albuterol (Salbutamol) 90 240→ 148 15

240→ 166 5

Cimetidine 100 253→ 159 10

253→ 95 25

Metformin 80 130→ 60 10

130→ 71 25

Ranitidine 110 315→ 176 15

315→ 130 25

×103

Counts vs. acquisition time (min)1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.05.5

6.0

6.5

7.0 1 1 2 2 3 3

Figure 1A. MRM extracted chromatogram for pharmaceuticals in Group 1. Three time segments were used in this chromatographic separation.

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×103

×102

×102

×103

×103

×103

0

1 1

0

1 1

0

1 1

0

1 1

0

1 1

0

1 1

Counts vs. acquisition time (min)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 2 0 21 2 2 23 24 2 5 26 27 28 29 30 31

×104

×102

×101

×103

×102

×103


0

1 1

0

1 1

0

1 1

0

1 1

0

1 1

0

1 1

0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5 9 9.5 10 10.5 11 11.5 12

Figure 1B. MRM extracted chromatogram for pharmaceuticals in Group 2. Only one transition shown. See Table 2B for compound identification.

Figure 1C. MRM extracted chromatogram for pharmaceuticals in Group 3. Only one transition shown. See Table 2C for compound identification.

479→ 462

465→ 430

458→ 441

445→ 410

445→ 428

427→

410

312→ 159.7

301→ 116.7

287→ 34.6

289→ 120.8

229→ 168.8

205→ 160.9

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Application to Wastewater Samples

To confirm the suitability of the method for analysis of real

samples, matrix-matched standards were analyzed in awastewater matrix from an effluent site, at eight concentra-

tions (0.1, 0.5, 1, 5, 10, 50, 100, and 500 ng/mL or ppb concen-

trations). Figure 2 shows an example standard curve for

acetaminophen in the wastewater matrix. In general, all com-

pounds gave linear results with excellent sensitivity over

three orders of magnitude, with r2 values of 0.99 or greater.

1 1

Cimetidine

Albuterol

Ranitidine

Metformin

253 & 159

& 95

240 & 166

& 148

315 & 176

& 130

130 & 71

& 60

×104

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

2.2

2.4

2.6

2.8

3

3.2


1 2 3 4 5 6 7 8 9 10 11 12 13 14

Figure 1D. MRM extracted chromatogram for pharmaceuticals in Group 4.

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Finally, a “blank” wastewater sample was analyzed and the

presence of two pharmaceuticals, carbamazepine and diphen-

hydramine, could be confirmed with two MRM transitions.

Figure 3 shows the ion ratios qualifying for these two com-

pounds in a wastewater extract. As shown in Figure 3 in the

two ion profiles, both pharmaceuticals were easily identifiedin this complex matrix due to the selectivity of the MRM tran-

sitions and instrument sensitivity.

Figure 2. Calibration curve for acetaminophen in a wastewater matrix using a seven-point curve from 0.1 to 100 ng/mL (ppb) using a linear fit with no origin

treatment.

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www.agilent.com/chem

Agilent shall not be liable for errors contained herein or

for incidental or consequential damages in connection

with the furnishing, performance, or use of this material.

Information, descriptions, and specifications in thispublication are subject to change without notice.

© Agilent Technologies, Inc., 2008Printed in the USASeptember 19, 20085989-9665EN

×104

×103

×103

×103

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.52 3

0

1

2

3

4

5

6

7 2 3

Counts vs. acquisition time (min) Counts vs. acquisition time (min)

14 15 16 17 18 19 20 21 22 23 24

0

1

2

3

4

5

6

7 2 3

0.0

0.5

1.0

1.5

2.0

2.5

2 3

14 15 16 17 18 19 20 21 22

Carbamazepine Diphenhydramine

237→ 194 256→ 167

237→ 179 256→ 152

Figure 3. MRM chromatograms of a wastewater sample for carbamazepine and diphenhydramine using two transitions.

Conclusions

The results of this study show that the Agilent 6410A Triple Quadrupole is a robust,

sensitive, and reliable instrument for the study of pharmaceuticals in water samples,

using high throughput methods. The Agilent 6410A Triple Quadrupole has been

shown to be a successful instrument for the implementation of EPA Method 1694.

References1. EPA Method 1694: Pharmaceuticals and personal care products in water, soil,

sediment, and biosolids by HPLC/MS/MS, December 2007, EPA-821-R-08-002.

Documents

5989-9665EN_EPA Method 1694